不同培养环境对PMBC中Treg和Th17细胞分化的影响

发布时间：2018-08-09 11:15

【摘要】：目的:研究不同培养环境对调节性T细胞(regulatory T cells,Treg)和Th17细胞分化的影响,探索能够逆转肿瘤微环境诱导的免疫耐受的手段。方法:克隆人白介素-6(IL-6)的基因,将其转入表达TGF-β的肿瘤细胞株中,并筛选稳转系;分别收集含或不含IL-6的肿瘤细胞培养上清,制备成条件培养基(Conditioned medium,CM),用于体外培养人外周血单核细胞(Peripheral blood mononuclear cells,PBMC),流式检测PBMC在不同培养环境中Treg和Th17细胞的变化情况。结果:RTPCR分析表明BEL-7402表达TGF-β的水平明显高于Hep G2,因此选择BEL-7402用于制备IL-6的稳转系;ELISA检测结果表明所制备的稳转系能够有效地表达IL-6;流式结果表明,由肿瘤细胞培养上清制备的CM能明显诱导T细胞分化为Treg,而在IL-6存在的情况下则能明显抑制Treg细胞的形成,并促进T细胞分化为Th17细胞。结论:肿瘤细胞培养上清促使PBMC中Treg比例增加;而在其中添加IL-6则能够降低Treg细胞比例,并增加Th17细胞的比例。
[Abstract]:Aim: to study the effects of different culture environments on the differentiation of regulatory T cell (regulatory T cells and Th17 cells, and to explore ways to reverse the immune tolerance induced by tumor microenvironment. Methods: the human interleukin-6 (IL-6) gene was cloned and transferred into the tumor cell line expressing TGF- 尾, and the stable transformation lines were screened, and the supernatants of tumor cells containing or without IL-6 were collected, respectively. A conditioned medium (Conditioned medium CM) was prepared for the culture of human peripheral blood monocytes (Peripheral blood mononuclear cells in vitro. The changes of Treg and Th17 cells in different culture environments were detected by flow cytometry. Results the level of TGF- 尾 expression in BEL-7402 was significantly higher than that in Hep G2. The results of Elisa showed that TGF- 尾 could express IL-6 effectively by using BEL-7402 to prepare IL-6, and flow cytometry showed that TGF- 尾 could express IL-6 effectively. The CM prepared from the supernatant of tumor cell culture could induce T cells to differentiate into Tregs, but in the presence of IL-6, it could inhibit the formation of Treg cells and promote the differentiation of T cells into Th17 cells. Conclusion: the supernatant of tumor cell culture can increase the proportion of Treg in PBMC, but the addition of IL-6 can decrease the proportion of Treg cells and increase the proportion of Th17 cells.
【作者单位】：广东药学院生命科学与生物制药学院/广东省生物技术候选药物研究重点实验室;
【基金】：国家自然科学基金(No.31100664,31300737,81303292) 广东省自然科学基金(No.10151022401000024,2014A030313586) 广东药学院师资队伍建设专项经费的资助
【分类号】：R392.12

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